Regulation of calcium signaling by the PKD2 gene product

描述（由申请人提供）：常染色体显性多囊肾病（ADPKD）是一种影响多器官和组织的通道病变。两个独立但相互作用的基因座pkd1和pkd2的突变是绝大多数ADPKD病例的原因。polycytin -2 （PC2）或PKD2属于瞬时受体电位（TRP）离子通道超家族。小鼠中pkd2的纯合缺失会导致肾脏和心脏缺陷导致的胚胎死亡，这可能是迄今为止所有已知TRP通道中最严重的表型之一。然而，对于诸如分子组装、门控和激活模式等基本特性的机制知之甚少。我们已经证明PKD2与其他通道亚基（如TRPC1）和辅助蛋白（如PKD1）以及哺乳动物中与蝶状体相关的双胍同源物（media1）相互作用。我们的功能数据显示PKD2在肾上皮细胞中形成EGF激活的质膜通道。在机制上，EGF以电压依赖的方式激活PKD2，并通过磷脂酶C (PLC)-gamma2和磷酸肌肽3-激酶（PI3K）的作用。新的初步数据表明，media1通过特异性抑制PKD2在负电位（超极化）而非正电位（去极化）下的活性，发挥了PKD2电压依赖性门的作用。media1的电压依赖性作用是由分子从负电位下的自抑制状态切换到正电位下的激活状态引起的。因此，我们假设PKD2形成了一种受体操作的通道复合物，其活性依赖于与其他通道（TRPC1）和辅助亚基（PKD1， media1和PLC-gamma2）的蛋白-蛋白相互作用。我们建议的具体目的是确定(1)PLC-gamma2及其底物磷脂酰肌醇-4,5-二磷酸（PIP2）， (2) media1和(3)TRPC1调节PKD2通道活性的机制。这些目标将主要通过PKD2中与PLC-gamma2、PIP2、media1和TRPC1相互作用结构域的生化鉴定及其电生理学功能表征来实现。几乎可以肯定的是，PKD2通道活性的扰动本身是ADPKD病理生理的主要原因之一。拟议的研究将帮助我们了解PKD2通道的功能和调控，并为自然发生的突变改变其活性的机制提供见解。

DESCRIPTION (provided by applicant): Autosomal dominant polycystic kidney disease (ADPKD) is a channelopathy affecting multiple organs and tissues. Mutations in two separate, but genetically interacting loci, pkd1 and pkd2, are responsible for the vast majority of all cases of ADPKD. Polycystin-2 (PC2) or PKD2 belongs to the transient receptor potential (TRP) superfamily of ion channels. Homozygous deletion of pkd2 in mice results in embryonic death due to kidney and heart defects representing perhaps one of the most severe phenotypes of all known TRP channels to date. However, little is known about the mechanisms underlying fundamental properties such as molecular assembly, gating, and modes of activation. We have shown that PKD2 physically interacts with other channel subunits such as TRPC1, and auxiliary proteins such as PKD1 and the mammalian homolog of diaphanous-related formin, mdia1. Our functional data show that PKD2 forms an EGF- activated plasma membrane channel in kidney epithelial cells. Mechanistically, EGF activates PKD2 in a voltage-dependent manner and through the action of phospholipase C (PLC)-gamma2 and phosphoinositide 3-kinase (PI3K). New preliminary data indicate that mdia1 functions as a voltage-dependent gate for PKD2 by specifically inhibiting its activity at negative (hyperpolarizing) but not positive (depolarizing) potentials. The voltage dependent action of mdia1 is caused by the molecular switching from its autoinhibited state at negative potentials to its activated state at positive potentials. We therefore hypothesize that PKD2 forms a receptor- operated channel complex whose activity is dependent on protein-protein interactions with other channels (TRPC1) and auxiliary subunits (PKD1, mdia1, and PLC-gamma2). The specific aims of our proposal are to determine the mechanism(s) by which (1) PLC-gamma2 and its substrate phosphatidylinositol-4,5- bisphosphate (PIP2), (2) mdia1, and (3) TRPC1 regulate PKD2 channel activity. These aims will be mainly accomplished by the biochemical identification of the interacting domains in PKD2 with PLC-gamma2, PIP2, mdia1, and TRPC1 and their functional characterization by electrophysiology. It is almost certain that perturbation of PKD2 channel activity per se is one of the major causes of ADPKD pathophysiology. The proposed studies will help us understand PKD2 channel function and regulation and provide insights into the mechanisms by which naturally occurring mutations alter its activity.